Gut bacterial aromatic amine production: aromatic amino acid decarboxylase and its effects on peripheral serotonin production

ABSTRACT Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.


Introduction
The effects of gut bacterial metabolites on host health have been demonstrated in rodent models. For example, acetic acid protects the host against pathogen infection 1 , butyric acid induces the colonic regulatory T cell differentiation, 2 deoxycholic acid increases liver cancer incidence, 3 lithocholic acid ameliorates inflammation in colitis, 4 and polyamines extend host longevity, 5,6 and maintain mucosal homeostasis. 7 The importance of gut bacterial metabolites is also recognized in humans has also been reported, e.g., de novo biosynthesis of vitamin K (menaquinones), which humans cannot biosynthesize, in gut. 8 Moreover, polyamines produced by gut bacteria contribute to atherosclerosis prevention. 9 Considering these findings, the regulation of gut bacterial metabolite production in the human is crucial for the maintenance of nutritional homeostasis and diseases prevention in humans. The molecular mechanism of gut bacterial metabolites production and their physiological function in the host have been gradually elucidated. For example, aromatic lactic acid produced by aromatic lactate dehydrogenase of Bifidobacterium affects the host immune system, 10 and isoallolithocholic acid produced by 5α-reductase and 3β-hydroxysteroid dehydrogenase of Parabacteroides merdae St3 through shows bactericidal activity against gram-positive pathogens. 11 However, our knowledge about the sources of gut bacterial metabolites and their production mechanism and physiological effects on host remains fragmentary.
Aromatic amines are among the compounds detected in the colon; 12,13 tryptamine, tyramine, and phenylethylamine (PEA) are typically known as the trace amines that can affect neurotransmission even in small amounts. 14,15 Diet and gut microbiota have been identified as sources of colonic luminal aromatic amines. Aromatic amines are found in fermented foods, 16 nuts, and citrus fruits 17 as well as in roasted coffee and cacao, where they are generated by Maillard-and Strecker reactions. [17][18][19] Several previous studies have reported that some gut bacteria possess aromatic amine-producing capabilities 20 and contribute to colonic aromatic amine level using germ-free animal models such as rats, chickens, and mice. [21][22][23][24] Previous in vitro studies have revealed that gut bacteria produce aromatic amines from aromatic amino acids through decarboxylation catalyzed by aromatic amino acid decarboxylase (AADC). 23,25 The kinetic parameters of AADC from several bacterial species, including gut bacteria (such as Enterococcus faecalis, Clostridium sporogenes, and Ruminococcus gnavus), have been determined using purified recombinant enzymes. 25,26 Almost all characterized AADC has shown relatively broad substrate specificity, 25,27 catalyzing the decarboxylation of not only proteinogenic aromatic amino acids (Phe, Tyr, and Trp), but also non-proteinogenic aromatic amino acids such as 3,4-dihydroxyphenylalanine (L-DOPA) [28][29][30][31] and 5-hydroxytryptophan. 27 Most previous studies on aromatic amino acid decarboxylation and aromatic amine production during the cultivation were focused on aromatic amine production from a non-proteinogenic aromatic amino acid L-DOPA. 29,32 Gut bacteria produce biogenic amines, including aromatic amines, from proteinogenic amino acids abundant in the diet, 33,34 such as tryptamine synthesis through the decarboxylation of tryptophan by gut bacteria. 25 However, few studies have elucidated at the genetic level how gut bacteria contribute to the production of trace amines from proteinogenic aromatic amino acids in the colon, and further research is needed to elucidate it.
PEA has been shown to induce efflux of neurotransmitters (dopamine, norepinephrine, and serotonin) and inhibit uptake of these neurotransmitters in human cell lines and in brain synaptosomes from mouse, juvenile rhesus, and tamarin. 35 PEA activates polymorphonuclear leukocytes and induces allergic reactions. 36 These physiological effects of PEA are mediated by trace amine-associated receptor 1 (TAAR1) activation. 35,36 Because TAAR1 is also expressed in the colonic epithelium, 37 PEA from gut bacteria is expected to exert physiological functions through TAAR1. 38 However, no studies have been conducted to investigate this possibility.
Serotonin (5-hydroxytryptamine) is a monoamine neurotransmitter distributed in the central and peripheral nervous systems, and plays different roles depending on location. Serotonin in the central nervous system is a neurotransmitter in the brain and affects sleep 39 and appetite, 40 whereas peripheral serotonin is a regulatory factor in different organs, regulating bone development, 41,42 immune response, 43 and brown adipose tissue thermogenesis. 44 Peripheral serotonin, which accounts for 90% of serotonin in the body, is produced by enterochromaffin (EC) cells in the gastrointestinal tract. 45 Bhattarai et al. demonstrated that Bacteroides thetaiotaomicron heterologously expressing aadc of R. gnavus produced tryptamine in the mouse gut, and the produced tryptamine increased anion and fluid secretion in the proximal colon via Serotonin receptor-4, one of the G protein-coupled receptors (GPCRs). 23 Gut bacteria modulate gastrointestinal motility and platelet function by promoting peripheral serotonin production from EC cells, mediated by tyramine and other gut bacterial metabolites. 24 Therefore, aromatic amines produced by gut bacteria have a significant effect on host physiology, by way of serotonin 24 or serotonin signaling pathways. 23,46 However, the serotoninmediated relationship between the host physiology and PEA, another natural aromatic amine, remains to be studied.
In this study, we identified five species of PEAproducing gut bacteria among 32 species of dominant human gut bacteria and verified that PEA production depends on aadc. The effects of aadc on colonic luminal aromatic amine and colonic serotonin production in the host were evaluated using mouse model. In addition, the PEA production by En. faecalis was successfully inhibited using established inhibitors. (GAM) 47 and evaluated polyamine biosynthesis and transport using this system. 48 Reanalysis of the high-performance liquid chromatography (HPLC) chromatograms obtained in the polyamine study revealed that Blautia hansenii, Clostridium asparagiforme, Tyzzerella nexilis, En. faecalis, and R. gnavus produced unidentified biogenic amine in the culture supernatant (Figure 1a and Supplementary Figure S1). The retention time of the unidentified biogenic amine did not correspond to that of polyamines (putrescine, cadaverine, spermidine, spermine, and agmatine) (Figure 1a). To identify the unidentified biogenic amine, we purified this compound using an ion exchange chromatography from the culture supernatant of T. nexilis. The MS/MS spectra of the purified biogenic amine corresponded to that of PEA ( Figure 1b). The retention time of the purified unidentified biogenic amine corresponded to PEA standard sample, in two different HPLC systems (Figure 1c and 1d). These results indicated that the unidentified biogenic amine is PEA.

Discovery of the PEA-producing bacteria in the most predominant species in the human indigenous gut microbiota
Five species of the human dominant gut bacteria (B. hansenii, C. asparagiforme, T. nexilis, En. faecalis, and R. gnavus) produced PEA in their culture supernatants. The PEA concentrations in the culture supernatants in stationary phase were 240 μM for B. hansenii, 74 μM for C. asparagiforme, 447 μM for T. nexilis, 2,572 μM for En. faecalis, and 1,317 μM for R. gnavus (Figure 1e).

Production of aromatic amines by PEA-producing gut bacteria in aromatic-amino-acids-defined medium
It was reported that PEA was biosynthesized from Phe in the reaction catalyzed by AADC, a pyridoxal-5′-phosphate-dependent decarboxylase. 25,27 AADC decarboxylates not only Phe, but also other proteinogenic aromatic amino acids (Tyr and Trp) 25,27 ( Figure 2a). The PEA-producing gut bacteria (B. hansenii, C. asparagiforme, T. nexilis, En. faecalis, and R. gnavus) possess the potential for the production of tyramine and tryptamine ( Figure 2a). As the concentrations of the substrates of AADC: Phe, Tyr, and Trp, in GAM were different from each other (Supplementary Table S1), the production ability of each aromatic amine could not be quantitatively compared based on the concentration of PEA, tyramine, and tryptamine in the culture supernatant. Therefore, an aromatic-amino-acid-defined (AAAD) medium, where the concentrations of aromatic amino acids were adjusted to 1 mM, was prepared (Supplementary Table S2). The concentrations of the corresponding aromatic amines, PEA, tyramine, and tryptamine, in the culture supernatant of PEAproducing gut bacteria were analyzed.
All tested PEA-producing gut bacteria could grow in the AAAD medium (Supplementary Figure S2) and PEA was detected in the culture supernatants of four species (B. hansenii, En. faecalis, R. gnavus, and T. nexilis) (Figure 2b to 2f). Aromatic amine concentrations at all measured cultivation times were analyzed by repeated measures one-way ANOVA (Supplementary Table S3). For those aromatic amines with significantly different concentrations, values at 0 h and 72 h after inoculation were subjected to a posthoc Tukey-Kramer test to verify whether they were significantly increased by the cultivation of the bacteria. Statistically significant aromatic amine production was observed, except for tyramine production by B. hasenii and tryptamine production by En. faecalis and C. asparagiforme (Figure 2b to 2d and  Supplementary Table S3). Production of PEA in the culture supernatants of PEA-producing gut bacteria grown in AAAD medium was reached the maximum at 72 h after inoculation; the concentrations were 13 μM for B. hansenii, 15 μM for C. asparagiforme, 208 μM for En. faecalis, 370 μM for R. gnavus, and 50 μM for T. nexilis, (Figure 2b to 2f). Then, one-way ANOVA and post-hoc Tukey-Kramer test (Figure 2b to 2f) were performed to compare the concentrations of the three aromatic amines in each species after 72 h of cultivation. In B. hansenii, the concentration of tryptamine was significantly higher than that of tyramine and PEA, with no significant difference between the concentrations of PEA and tyramine ( Figure 2b). In C. asparagiforme, the concentration of tyramine was significantly higher than that of PEA and tryptamine, with no significant difference between the concentrations of PEA and tryptamine ( Figure 2c). In En. faecalis, the concentration of tyramine was significantly higher than that of PEA and tryptamine, with no significant difference between the concentrations of PEA and tryptamine (Figure 2d). Although there was no significant difference between the production of PEA and tryptamine in En. faecalis, the p-value obtained in the Tukey-Kramer test was   0.109, indicating a tendency toward higher PEA production than tryptamine (Figure 2d). In R. gnavus, the concentration of tryptamine and PEA were significantly higher than that of tyramine, with no significant difference between the concentrations of PEA and tryptamine (Figure 2e). In T. nexilis, the concentration of tryptamine was significantly higher than that of PEA and tyramine, with no significant difference between the concentrations of PEA and tyramine ( Figure 2f).
To determine the productivity of aromatic amine by AADC and AADC homologs of PEA-producing gut bacteria, the genes or its homologs encoding AADCs: aadc Rg , aadc Ef , aadc Bh , aadc Ca , and aadc Tn were cloned into overexpression vectors and introduced into E. coli and the concentration of aromatic amines in the culture supernatants of E. coli strains were measured.
E. coli harboring the empty vector (YS297) did not produce any of the aromatic amines (Figure 3a), whereas the E. coli strains heterologously expressing the aadc and aadc homologs produced more than 600 μM PEA, 800 μM tryptamine, and 300 μM tyramine at 48 h after inoculation (Figures 3b to 3f), confirming that the production of aromatic amines was due to the heterologous expression of the aadc and aadc homologs. Aromatic amine concentrations at all measured cultivation times were analyzed by repeated measures one-way ANOVA (Supplementary Table S4). For the aromatic amines with significantly different concentrations, values at 0 h and 48 h after inoculation were subjected to a post-hoc Tukey-Kramer test to verify whether they were significantly increased by the cultivation of the bacteria. Statistically significant aromatic amine production was observed, except for tryptamine production by YS317 harboring aadc Ef (Figure 3d and Supplementary Table S4). One-way ANOVA and post-hoc Tukey-Kramer test (Figure 3b to 3f) were performed to compare the concentrations of the three aromatic amines in the culture supernatant of each E. coli strain after 48 h of cultivation. In YS389 harboring aadc Bh , the concentration of tryptamine and PEA was significantly higher than that of tyramine ( Figure 3b). In contrast to the results obtained from B. hansenii cultured in AAAD medium (Figure 2b), the results obtained from YS389 showed a significantly higher concentration of PEA than tyramine at the end of cultivation ( Figure 3b). In YS300 harboring aadc Ca , the concentration of tyramine was significantly higher than that of PEA and tryptamine ( Figure 3c). Similar results were obtained when C. asparagiforme was cultured in AAAD medium ( Figure 2c). YS389, YS300, YS298, and YS299 harboring aadc Bh (Figure 3b), aadc Ca (Figure 3c), aadc Rg (Figure 3e), and aadc Tn (Figure 3f), respectively, produced tryptamine, whereas YS317 harboring aadc Ef did not produce tryptamine during the cultivation period. In contrast to the culture supernatant of En. faecalis in AAAD medium (Figure 2d), the culture supernatant of YS317 harboring aadc Ef showed no significant difference in the final concentrations of PEA and tyramine, although the concentration of PEA reached its maximum after tyramine ( Figure 3d). In YS298 harboring aadc Rg , the concentration of PEA was significantly higher than that of tryptamine ( Figure 3e). The concentrations of tryptamine and tyramine were significantly different in R. gnavus cultivated in AAAD medium ( Figure 2e) but not in YS298 (Figure 3e). In contrast to T. nexilis cultivated in AAAD medium (Figure 2f), the culture supernatant of YS299 harboring aadc Tn contained a significantly higher concentration of PEA and tyramine than tryptamine ( Figure 3f).

Correlation of gut bacterial aadc copy number and PEA production ability in human feces
Heterologous expression experiments indicated that aadc candidates: aadc Bh , aadc Ca , aadc Tn , were involved in PEA production ( Figure 3b to 3f), in addition to aadc Ef and aadc Rg, whose role in PEA production was already reported. However, it was still unclear whether aadc of PEA-producing gut bacteria produce PEA in the human colonic lumen. Therefore, the correlation between the copy number of aadc Ef , aadc Rg , aadc Bh , and aadc Tn and the amount of PEA produced when incubated with 1 mM Phe was analyzed, using human fecal samples. C. asparagiforme produced 254 μM of tyramine in AAAD medium; however, PEA production was extremely low (15 μM, Figure 2c), and therefore, we excluded aadc Ca from the analysis.
The concentrations of PEA produced in the fecal culture ranged from 0 to 2.6 μM (Figure 4a and 4b). aadc Tn was detected in 44% of donors (4/9); aadc Rg, in 88% (8/9), while aadc Ef and aadc Bh were not detected by the qPCR assay. A significant correlation was observed between PEA production and aadc Rg (r = 0.8216 and p= 0.0066, Figure 4b), but not between PEA production and aadc Tn (r = 0.5000 and p= 0.1704, Figure 4a).

Influence of gut bacterial aadc on colonic aromatic amine production in colon and serotonin level
We hypothesized that colonic luminal PEA, as with tyramine, 24 induces serotonin production from EC cells via TAAR1. To confirm this hypothesis in One-way ANOVA: p < 0.001 One-way ANOVA: p < 0.001 One-way ANOVA: p < 0.05 One-way ANOVA: p < 0.05 One-way ANOVA: p < 0.001 One-way ANOVA: not calculated experiments with BLAB/cCrSlc mice, we purchased and used the colons of BLAB/cCrSlc mice to evaluate Taar1 expression in colon. RNA was extracted from the purchased colons of BALB/cCrSlc mice, PCR was performed using the reverse transcription product as a template with Taar1-specific primers, 50 and the PCR product was sequenced. As a result, the expression of Taar1 in the BALB/ cCrSlc mouse colon was confirmed (Supplementary Figure S3). Next, to analyze the effects of indigenous bacterial PEA production in the intestinal lumen on the colonic serotonin production in the host at gut bacterial gene level, we selected En. faecalis, which can be genetically engineered, among the PEA-producing gut bacteria and generated aadc-deletion (SK981) and complementation (SK982) strains of En. faecalis. The production of aromatic amines was completely lost by the deletion of aadc, as previously described 29 and the production of aromatic amines was recovered by the complementation of aadc (Figure 5a). The colonic serotonin levels of mice colonized with wildtype En. faecalis (SK947), Δaadc (SK981), or aadccomplemented strain (SK982) were measured. A schematic overview of the mice experiment is shown in Figure 5b. The number of En. faecalis was almost the same in the feces of mice colonized with wild type, ∆aadc, and aadc-complemented En. faecalis, suggesting that the colonization efficiency was not influenced by aadc deletion or complementation ( Figure 5c). The fecal aadc copy number was significantly higher in mice colonized with aadccomplemented strain than in those colonized with wild-type En. faecalis. The fecal aadc copy number of mice colonized with ∆aadc En. faecalis was in the order of 1/3,000, compared to that of mice colonized with wild type En. faecalis (Figure 5c). Serotonin levels were significantly higher in the colon tissue of mice colonized with aadccomplemented strain than those colonized with wild-type En. faecalis or ∆aadc En. faecalis. However, no significant difference in the serotonin levels in the colon tissue was observed between mice colonized with wild-type En. faecalis and mice colonized with ∆aadc En. faecalis ( Figure 5d). These data suggest that a high copy number of aadc (~10 10 copy number/g of feces), which was also observed in some samples during our human fecal assays (Figure 4), induces colonic serotonin production (Figure 5c and 5d). However, PEA was not detected in the feces and the cecal contents of any of the mice. A small amount of tyramine was detected in the feces of mice colonized with wild-type and aadc-complemented En. faecalis, while no tyramine was detected in the feces of mice colonized with ∆aadc En. faecalis (Supplementary Figure S4A). These results indicate that gut bacterial aadc contributes to aromatic . PEA production in feces is associated with aadc of R. gnavus. Nine human feces were separately incubated with or without 1 mM Phe. PEA production was calculated from the difference between PEA concentration when incubated with 1 mM Phe and that without 1 mM Phe. Copy number of aadc of T. nexilis and R. gnavus were determined using qPCR. (a) Correlation between PEA production and copy number of aadc of T. nexilis. (b) Correlation between PEA production and copy number of aadc of R. gnavus. Spearman's rank correlation test was used for the correlation analysis (r = correlation coefficient). The copy number of the aadc gene in samples where the aadc gene was below the detection limit in our system was set as 10 6 copies/g of feces. Donors are distinguished by color; the same color in (a) and (b) indicates the same donor.   Strains were cultured at 37°C in GAM with chloramphenicol (10 μg/mL) for 72 h in an anaerobic chamber. Aromatic amine concentration in the culture supernatants was quantified using HPLC. Data represent the mean ± SD of three individual experiments. One-way ANOVA was performed to assess for significant differences in the aromatic amine concentrations between the groups, and the p-values for one-way ANOVA are indicated. Statistical significance between the strains was further analyzed by Tukey-Kramer test. The groups indicated by different letters were statistically different to each other, and the amine production in the host colonic lumen. However, no significant correlation between the fecal tyramine amounts and colonic serotonin amounts was observed in the groups (Supplementary Figure S4B).

Human AADC inhibitor decreases PEA production in En. faecalis
Several human AADC inhibitors have been clinically used for the treatment of Parkinson's disease, 51 and these inhibitors may be candidates for drug repositioning. We evaluated whether human AADC inhibitors (carbidopa, methyldopa, and benserazide [ Figure 6a]) could inhibit PEA production of En. faecalis and R. gnavus, which produced PEA in vitro (Figure 2d and 2e). Severe growth deficiency was not observed with any tested AADC inhibitors (Supplementary Figure S5A and S5B). PEA production in En. faecalis was strongly inhibited by carbidopa and benserazide, however methyldopa did not inhibit PEA production (Figure 6b and Supplementary Figure S5C). Surprisingly, tyramine concentration in the culture supernatant of En. faecalis did not change when treated with carbidopa and benserazide (Figure 6c and Supplementary Figure S5D). None of the tested human AADC inhibitors inhibited PEA production by R. gnavus (Figure 6d and Supplementary Figure  S5E). Although carbidopa and benserazide significantly reduced tyramine production by R. gnavus (Supplementary Figure S5F), they also significantly reduced R. gnavus growth (Supplementary Figure  S5B). Therefore, the tested inhibitors exerted no significant effects after the tyramine concentration of R. gnavus was normalized to bacterial growth (OD 600 ) (Figure 6e).

Discussion
Gut microbiota produce various metabolites in the colonic lumen, affecting the host physiology. 52 Identification of gut bacterial species and genes responsible for metabolite production is essential for the optimization of the state of the intestinal environment. Some molecular mechanisms of metabolite production in the colonic lumen have been established at the genetic level, 53-55 however, even for well-known metabolites, there are unidentified mechanisms modulating the production. Few studies have investigated between intestinal bacterial genes and the host physiology. In this study, we identified five PEA-producing bacteria from the GAM-culturable human dominant gut bacteria ( Figure 1e) and revealed that aadc is indispensable for the production of PEA (Figures 3b-3f, and 5a). Our results suggested that gut bacterial AADC and its reaction product, PEA, participate in the peripheral serotonin production in the host. Furthermore, fecal culture and mouse experiments indicated that gut bacterial aadc contributes to aromatic amine production in the colon (Figure 4b and Supplementary Figure S4A). AADC of R. gnavus was recently identified as a Trp decarboxylase, and studies using the recombinant AADC indicated that the catalytic efficiency (k cat /K m ) for Trp is 1,000-fold higher than that for Phe. 25 However, in our experiment using the AAAD medium containing 1 mM of Trp, Tyr, and Phe, R. gnavus produced statistically p-values between the groups indicated by the different letters are shown in each panel. (b) The feeding schedule for mice. Six-weeksold female BALB/cCrSlc mice were given antibiotics in drinking water for two weeks to remove the indigenous bacteria. Mice were fed a standard diet for 13 d, then a Phe-rich diet for 5-days. En. faecalis (WT, Δaadc, aadc complementation) (1 × 10 8 cfu) was inoculated into mice on day 15, indicated by the red arrow (1 day after stopping antibiotics treatment). (c) En. faecalis colonization of mouse colon confirmed using qPCR. Statistical analysis was performed using Steel-Dwass test. No significant differences were observed between the groups, for total bacterial 16S rRNA gene and En. faecalis 16S rRNA gene. Two independent experiments were performed (n = 4 and 6 in each group, respectively), and data is shown in box plots with the median ± interquartile range. Statistical significance for each gene copy number between the groups was assessed by the Kruskal-Wallis test post-hoc Steel-Dwass test. significantly more tryptamine than PEA, but the difference was 1.2-fold, which was much smaller than the difference expected from k cat /K m value (Figure 2e). B. hansenii (Figure 2b) and T. nexilis (Figure 2f), which were considered tryptamineproducing gut bacteria 25 produced not only tryptamine, but also PEA and/or tyramine in the AAAD medium. Aromatic amine productivity of E. coli transformants expressing each aadc from PEAproducing gut bacteria in M9AAA-medium were partially but not entirely consistent with the PEAproducing gut bacteria in AAAD medium (Figures 2b to 2f and 3b to 3f). These differences were hypothesized to be due to the different abilities of E. coli and PEA-producing gut bacteria to uptake aromatic amino acids or to release aromatic amines. These results suggested that the production of metabolites secreted by bacterial cells is influenced not only by the activity of the enzymes, but also by that of transporters. A significant correlation was observed between aadc Rg and the production of PEA in fecal culture (Figure 4b). Therefore, our results suggest that PEA production was not a side reaction, but rather the main reaction catalyzed by AADC in the intestinal lumen. On the other hand, no significant correlation was observed between aadc Tn and the PEA production ( Figure 4b). This could possibly be because of the lower number of the fecal samples possessing aadc Tn . In addition, in vitro analysis showed that R. gnavus produced more PEA than T. nexilis (Figure 2e and 2f), suggesting that the contribution of aadc Rg is significant compared to that of aadc Tn, in the PEA production in colonic lumen. We also observed that while possessing the same level of aadc Rg in the feces, the fecal samples differ in PEA production (Figure 4b). Gut bacteria reductively metabolize aromatic amino acids to aromatic lactate, aromatic acrylate, and aromatic propionate by aromatic lactate dehydrogenase, phenyl lactate dehydrogenase, phenyl lactate dehydratase, or acyl-CoA dehydrogenase after deamination. 10,55 Therefore, we speculate that the amount of the genes related to reductive aromatic amino acid metabolism pathway affects PEA production.
Peripheral serotonin is produced by EC cells 56 and its production heavily relies on gut bacteria. 24 Physiologically active metabolites derived from gut bacteria, such as short-chain fatty acids (acetic acid and butyric acid), 57 and deoxycholate 24 stimulate peripheral serotonin production. Intrarectally injected tyramine induces peripheral serotonin secretion in mouse. 24 EC cells express a variety of receptors, including TAAR1; 58 like tyramine, PEA is also a ligand of TAAR1. 35 Colonic Taar1 expression has been reported in humans, 37 and BALB/cCrSlc mice of the same strain as used in the experiment to assess the effect of gut bacterial PEA on the colonic serotonin, was also expressing Taar1 in the colon (Supplementary Figure S3). Furthermore, activation of TAAR1 induced serotonin production in a cell model. 35 In this study, colonic serotonin levels of mice colonized with aadc-complemented En. faecalis were higher than those in mice colonized with wild-type En. faecalis and Δaadc En. faecalis (Figure 4d). There are two possible sources of phenylalanine in the mouse colon: diet and metabolic activity of gut bacteria. Phe, a substrate for AADC, has been detected in mice colon and its amount increase in response to feeding a high-fat diet. 59 Therefore, it is possible that PEA is generated in the murine colonic lumen by the reaction catalyzed by AADC. However, in our experiments, PEA was detected neither in the feces nor in the cecal contents of mice. A possible reason for this is that PEA produced by gut bacteria in the intestinal tract after inducing serotonin production in mice was rapidly degraded by a reaction catalyzed by monoamine oxidase-B (MAO-B) 60 and was undetectable by the time of analysis. Actually, the expression of Mao-b in the colon of BALB/cCrSlc mice of the same strain as used in the experiment to assess the effect of gut bacterial PEA on the colonic serotonin was confirmed by PCR (Supplementary Figure S3). The reason why tyramine, but not PEA, was detected (Supplementary Figure S4A) in the colonic lumen could be that the K m of MAO-B, which reportedly is expressed in the intestinal tract, 24 to tyramine is described to be 290-fold higher than that to PEA. 61 Therefore, it is likely that only PEA was preferentially degraded by MAO-B. In the present study, a maximum of 41.8 pmol of tyramine per mg of feces was detected (Supplementary Figure S4A), but tyramine concentration in the feces did not correlate significantly with serotonin production in the intestinal tract (Supplementary Figure S4B). On the contrary, Yano et al. demonstrated that injection of about 20 μmol of tyramine into the mouse colonic lumen activated colonic serotonin production. 61 Given a mouse fecal mass of 100 to 200 mg, 62,63 the amount of tyramine detected in the colon in the present study was 4.2 to 8.4 nmol, much less than the dose given by Yano et al. 24 This could explain why tyramine amounts in the intestinal lumen did not correlate with serotonin production in the intestinal tract; thus, this study does not exclude the theory that tyramine promotes serotonin production 24 in the colon.
Peripheral serotonin is an important regulator and is implicated in several diseases, such as osteoporosis, 64 diarrhea-predominant irritable bowel syndrome, 65 celiac disease, 66 inflammatory bowel disease, 67 and obesity. 68 Marketed drugs: Carbidopa and benserazide, developed as human AADC inhibitor, significantly inhibited PEA production in En. faecalis (Figure 6b). However, none of these inhibitors inhibited tyramine production by En. faecalis and PEA and tyramine production by R. gnavus (Figure 6c, 6d, and 6e). AADC Ef is capable of decarboxylating L-DOPA, while AADC Rg cannot. 29 The human AADC inhibitor is an analogue of L-DOPA, and therefore likely fits into the substrate pocket of AADC Ef to inhibit PEA production. However, the low affinity of AADC Rg for L-DOPA might have abrogated the inhibitory effect. (S) -α-fluoromethyl tryptophan inhibits AADC of R. gnavus, 25 while (S)-α-fluoromethyl tyrosine inhibits AADC of En. faecalis . Therefore, (S)-α-fluoromethyl derivatives, such as (S)-αfluoromethyl phenylalanine may be promising as inhibitors of PEA synthesis by AADC of gut bacteria.
Here, we showed that gut bacterial AADC contributes colonic serotonin production, and that two commercial AADC inhibitors can inhibit PEA production by En. faecalis. However, our study had some limitations. First, we used BALB/cCrSlc mice as a model. This strain possesses a mutation in the tryptophan hydroxylase-2 gene, which is responsible for central serotonin biosynthesis. 69,70 BALB/cCrSlc mice has not been reported to exhibit a mutation in tryptophan hydroxylase-1, which is responsible for the peripheral serotonin biosynthesis evaluated in the present study. The determined colonic serotonin level (0.49 to 6.8 μg/g of colonic tissue) was comparable to that in previous studies using C57BL/6 mice. 71 However, other models are needed to assess PEA effects on serotonin, including in the central nervous system. Second, we evaluated the effects of human AADC inhibitors on gut bacterial aromatic amine production with 1.5 mM. This dosage has technical and toxicological limitations, e.g., to reach 1.5 mM of carbidopa in the intestinal tract, a minimum of 1 g must be ingested. 72 To solve this problem, it is necessary to develop efficient drug delivery methods to the intestinal tract; for example, the administration of carbidopa in enteric capsules and the development of effective gut bacterial AADC inhibitors that are not absorbed in the intestine should be considered. In the future, selective and efficient inhibition of gut bacterial AADC could lead to the prevention and treatment of diseases involving peripheral serotonin.

Conclusion
The present study presented five PEA-producing gut bacteria species and determined that aromatic amine production from proteinogenic aromatic amino acids depends on AADC activity. Human fecal culture experiments revealed a significant positive correlation between aadc of R. gnavus and PEA production, suggesting that R. gnavus contributes to PEA production in the human colonic lumen. Furthermore, gut bacterial aadc activity upregulated colonic serotonin levels in a mice model treated with genetically modified En. faecalis. Finally, we revealed that the human AADC inhibitors carbidopa and benserazide prevented PEA production by En. faecalis. This study shows that AADC in gut bacteria may be a potential target in the prevention and treatment of diseases involving peripheral serotonin.

Bacterial strains
The strains used in this study are listed in Table 1. Bacteria were obtained from the Japan Collection of Microorganisms (JCM), the American Type Culture Collection (ATCC), and the German Collection of Microorganisms and Cultures (DSMZ).

High-performance liquid chromatography (HPLC)
Quantification of aromatic amines and aromatic amino acids were carried out using HPLC. PEA was quantified by the post-column labeling method as describe previously. 82 Briefly, PEA was separated using a cation-exchange column (#2619PH, Hitachi, Tokyo, Japan) in normal-phase mode, and derivatized with o-phthalaldehyde and detected using fluorescence detector (λ ex : 340 nm and λ em : 435 nm). Tyramine and tryptamine were separated in reversephase mode using a Discovery HS-F5 column (4.6 × 250 mm, 5 μm, SUPECLCO, Bellefonte, PA, USA) at 35°C. The elution was carried out at a flow rate of 0.4 mL/min with 10 mM ammonium formate (pH 3.0) and acetonitrile using the following gradient program: the concentration of acetonitrile was linearly increased from 3 to 27% during 0-22 min, and linearly increased from 27 to 66% during 22-80 min, increased to 100% during 80-81 min, maintained at 100% during 81-86 min, returned to 3% during 86-87 min, and maintained at 3% during 87-102 min. In this analytical system, the retention time for Tyr, tyramine, Trp, and tryptamine were 18.5, 28, 29.3, and 51 min, respectively. The elution was monitored based on the fluorescence with excitation at 280 nm and emission at 325 nm, using a Waters 2475 Multiwavelength Fluorescence detector (Waters, Milford, MA, USA). Phe concentration was quantified using HPLC equipped with a cation-exchange column (#2619, Hitachi). The temperature of the column oven was maintained at 70°C. Buffer-A (23 mM sodium citrate, 96.8 mM sodium chloride, 84.7 mM citric acid, 13% ethanol, and 0.0001% caprylic acid), buffer-B (26.3 mM sodium citrate, 121 mM sodium chloride, 105 mM citric acid, 0.02% ethanol, and 0.0001% caprylic acid), buffer-C (90 mM sodium citrate, 930 mM sodium chloride, 104 mM citric acid, and 0.0001% caprylic acid), and buffer-D (200 mM sodium hydroxide, 10% ethanol, and 0.0001% caprylic acid) were used as the mobile phase, at a flow rate of 0.4 mL/min. The gradient program was as follows: buffer-A was kept at 100% during 0-13 min. The buffer was changed to buffer-B and the proportion was kept at 100% from 13-18 min. From 18-26 min, buffer-B was linearly decreased from 100 to 75% and buffer-C was linearly increased from 0 to 25%. Buffer-C was linearly increased to 100% during 26-56 min and kept at 100% during 56-67 min. The buffer was changed to buffer-D and the proportion of buffer-D was kept at 100% during 67-74 min. The buffer was changed to buffer-A and the proportion of buffer-A was kept at 100% during 74-105 min. Detection of Phe was carried out using the same method as that for PEA. A standard sample was always analyzed within each batch to quantify aromatic amines and aromatic amino acids in the sample. Aromatic amine and aromatic amino acid species in the samples were identified by comparison with the retention times of the standards, and each compound concentration was determined using a standard curve generated from the peak areas of the standards at known concentrations.

Purification of unidentified biogenic amine from culture supernatant of T. nexilis
The unidentified biogenic amine was purified from culture supernatant of T. nexilis using a cation exchange resin (Dowex 50 W X8, [H + -form; 100-200 mm], FUJIFILM Wako Pure Chemicals). Ten milliliters of resin was packed into a column.
T. nexilis was anaerobically pre-cultured overnight in 1 mL of GAM at 37°C. Pre-cultures (500 μL) were inoculated into 500 mL of GAM and cultured overnight at 37°C in an anaerobic chamber (INVIVO2 400; Ruskinn Technology, Bridgend, UK) until stationary phase. 47 The culture was centrifuged (12,680 × g, 4°C, 20 min), and the supernatant was applied to the resin-packed column. After loading, the unidentified biogenic amine was purified using the following procedures: the column was washed with 400 mL of deionized water. Polyamines (spermidine, cadaverine, and spermine) were eluted with 120 mL of 1 M HCl. Residual HCl was removed from the column by washing the column with 200 mL of deionized water. Agmatine was eluted with 115 mL of 500 mM of NH 3 and the column was washed with 100 mL of deionized water. The unidentified biogenic amine was eluted with 100 mM HCl. The eluted unidentified amine was again absorbed onto the same column, washed with 2 L of deionized water, and then eluted by increasing the NH 3 concentration in steps of 2, 4, and 8 M in 80 mL increments. Every 1 mL was recovered, and the concentration and purity of the unidentified biogenic amine were tested using HPLC. The fraction eluted with 8 M NH 3 contained the highest amount of the unidentified biogenic amine, and was therefore subjected to further analysis.

Determination of molecular mass of the purified unidentified biogenic amine using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)
The mass of the purified product was determined using a 3200 QTRAP MS system (Applied Biosystems, Foster City, CA, USA). The sample was directly injected to the electrospray ionization-MS detector in scan mode with positive ionization. The molecular ion peak with m/z of 122 was further analyzed by MS/MS fragmentation. The purified sample was separated using an Agilent HPLC system (Agilent) as follows: column, TSKgel ODS-80Ts (4.6 × 250 mm 2 , 5 μm particles; Tosoh); solvent system, A: 0.1% acetic acid in water, B: 0.1% acetic acid in acetonitrile; gradient modes: 90% A (0-5 min), 90-60% A (5-20 min), and 10% A (20-30 min); flow rate, 0.5 mL/min at 40°C.
The separated sample was analyzed by LC-MS/MS, using select ion mode at m/z = 122. Standard PEA was used for comparison. The biogenic amine was confirmed as PEA by comparing its retention time and daughter ions with that of standard PEA in LC-MS/MS analysis.
Aromatic amine production profile of PEA-producing gut bacteria PEA-producing gut bacteria were anaerobically precultured in GAM at 37°C for 18 h in an anaerobic chamber (INVIVO2 400). After pre-cultivation, cells in the stationary phase 47 were washed and resuspended with 30 mL of the AAAD medium (Supplementary Table S2) at an initial OD 600 of 0.03 and cultured at 37°C in an anaerobic chamber. One milliliter of the culture was collected at the indicated times and centrifuged (18,900 × g, 4°C, 10 min) to obtain the culture supernatant. The culture supernatant was filtrated using a Cosmonice filter W (Nacalai Tesque) after protein precipitation by trichloroacetic acid, as described previously. 82 The filtrated sample was analyzed using HPLC.

Aromatic amine production profile of En. faecalis and R. gnavus in the presence of human AADC inhibitors
Human AADC inhibitors: carbidopa, 83 methyldopa, 84 and benserazide 85 were dissolved in MilliQ water and added to the AAAD medium at a final concentration of 1.5 mM. The other culture conditions were the same as described above. Following 24 h cultivation in the presence of inhibitors, PEA and tyramine concentrations in the culture supernatants and OD 600 were measured.

Plasmid construction
Cells of B. hansenii, C. asparagiforme, T. nexilis, En. faecalis, and R. gnavus in 0.5 mL of overnight culture, were centrifuged and stored at −20°C until the genomic DNA (gDNA) extraction. Cells of T. nexilis and R. gnavus were suspended in 100 μL of TE buffer and disrupted using zirconia beads (Thermo Fisher Scientific, Waltham, MA, USA) in a SHAKE MASTER ver. 1.2 (Bio Medical Science, Tokyo, Japan). The disrupted cells were centrifuged (21,500 × g, 4°C, 10 min) and gDNA in the resulting supernatant fractions were used as templates in PCR reactions. B. hansenii and C. asparagiforme gDNA were extracted using the phenol-chloroform methods. 47  Plasmids used for generating a standard curve of 16S rRNA genes and aadc Ef in qPCR were constructed as follows: 16S rRNA gene and aadc Ef were amplified from gDNA of En. faecalis using the primers listed in Table 1. Amplified DNA was cloned into SmaI site of pUC19 using DNA ligation kit Mighty Mix (TaKaRa Bio).

Heterologous expression of aadc in Escherichia coli
Escherichia coli BL21 (DE3) strains harboring aadc expression plasmids or an empty plasmid were pre-cultured in 5 mL of Luria-Bertani medium containing 75 μg/mL spectinomycin in 100 mL Erlenmeyer flask, at 37°C with reciprocal shaking at 140 rpm for 17 h. M9 medium 86 containing 0.2% of glucose and 1 mM of aromatic amino acids: Phe, Tyr, and Trp (M9AAA-medium) was used for the main culture. Pre-cultured cells were washed with M9 medium containing 0.2% glucose and suspended in M9AAA-medium. The suspension was inoculated into 10 mL of M9AAA-medium, supplemented with 10 μM isopropyl β-D-thiogalactopyranoside in a 100 mL Erlenmeyer flask at an initial OD 600 of 0.03 and cultured at 37°C with reciprocal shaking at 140 rpm. An aliquot of the culture was collected every 6 h and centrifuged for 10 min at 21,500 × g at 4°C. The resulting culture supernatant was treated with 10% (w/v) trichloroacetic acid, and was subjected to HPLC analysis, as previously reported. 87

Generation of deletion mutant of aadc in En. faecalis V583
The aadc gene of En. faecalis V583 was deleted using pLT06 as described previously. 53 Primers and plasmids used for aadc deletion and complementation are listed in Table 1. Complementation plasmid pLZ12-aadc + was constructed as follows: aadc and its upstream 500 bp were amplified using PCR with PrimeSTAR Max DNA polymerase (TaKaRa Bio) with C_ddc+0.5K_F_pLZ_Bam and C_ddc+0.5K_R_pLZ_Bam as primers. The product was cloned into BamHI site of pLZ12. The resulting plasmid was introduced into En. faecalis by electroporation, as described previously. 53

Conversion of phenylalanine to phenylethylamine by human fecal sample
Fecal samples from nine healthy Japanese donors (eight male and one female; age: 21.8 ± 10.2, range 4-40 years) were analyzed. Feces were collected using stool collecting kit (LSI medience Co., Tokyo, Japan) and stored under anaerobic condition using Anaero Pack system (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at −80°C until use. Fecal samples were suspended in a 4-fold volume of phosphate-buffered saline (PBS) or PBS containing 1 mM of Phe and were incubated anaerobically at 37°C. After 8 h incubation, the culture supernatants were harvested by centrifugation (21,500 × g, 4°C, 10 min) and subjected to HPLC analysis, as described in the "Aromatic amine producing profile of PEA-producing gut bacteria" section. The net PEA production was calculated by subtracting PEA amount formed in the absence of Phe from that formed in the presence of 1 mM Phe in the fecal suspension.

Extraction of gDNA from fecal samples
Fecal samples (10-20 mg) were suspended in 95 μL of TE buffer. Five microliters of 300 mg/mL lysozyme (Sigma-aldrich, MO. USA) and 11 μL of 1,000 U/μL achromopeptidase (FUJIFILM Wako Pure Chemicals) were added to the suspension and incubated for 30 min at 37°C. Twelve microliters of 20% sodium dodecyl sulfate solution was added to the suspension and incubated at 60°C for 20 min. 88 The bacterial DNA was extracted from the incubated mixture using QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany).

Quantification of DNA copy numbers in fecal sample
The copy number of the targeted gene was determined by quantitative PCR (qPCR) using a thermal cycler (StepOne Real-time PCR system, Applied Biosystems) and TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Bio). Primers used for qPCR are listed in Table 1. The Reaction mixture (20 μL) consisted of 10 μL of 2× TB Green II mix, 9.2 ng of gDNA, and 0.7 μM (for aadc gene) or 0.35 μM (for 16S rDNA) of primers. PCR cycling conditions for the amplification of aadc Tn were 95°C for 30s, followed by 40 cycles of 95°C for 5 s and 64°C for 45s. PCR cycling conditions for the amplification of the other genes were 95°C for 30s, followed by 40 cycles of 95°C for 5 s and 60°C for 1 min. Gene copy numbers were calculated based on the standard curve generated using varying concentrations of gDNA or plasmids containing the target gene.

Animal experiments
Six-week-old female BALB/cCrSlc mice were purchased from Japan SLC (Hamamatsu, Japan). All mice were maintained in a 12 h light-dark cycle and housed in group cages with 2-3 animals per cage with free access to water and diet and bred based on the regulations regarding the protection of laboratory animals at the Kanazawa University. All animal experiments were performed according to the Guideline for the Care and Use of Laboratory Animals at Kanazawa University (Approval number: AP-163778). Mice were fed a standard diet (CE-2, CLEA Japan Inc., Tokyo, Japan) for 13 days. Tyr was removed from the diet and Phe content was increased from 0.87% (w/w) in the L-Amino Acid Defined AIN-93 G (Dyets Inc., Bethlehem, PA, USA) (Supplementary Table S5) to 8.7% (w/w) in the Phe-rich diet (#511379, Dyets Inc.) (Supplementary Table S5). The mice were fed a Phe-rich diet from for 5 d, beginning on day 13 (Figure 4b). Drinking water and the diet were sterilized by autoclave or γ-irradiation, respectively. Antibiotics (0.25 mg/mL of doripenem and 0.5 mg/mL of vancomycin) were provided ad libitum in the drinking water 89 for 14 days until one day before En. faecalis administration.
The En. faecalis strains, WT, Δaadc, and aadc complemented En. faecalis were grown anaerobically in GAM containing 10 μg/mL chloramphenicol at 37°C for 12 h. Cells were collected from 1 mL of culture by centrifugation (6,000 × g, 25°C, 5 min) and washed twice with 1 mL of PBS containing 10 μg/mL chloramphenicol. Cells were suspended in 5 mL of PBS and the colony forming unit (cfu) was measured on GAM agar plate containing 10 μg/ mL chloramphenicol. Cells were prepared to 1 × 10 8 cfu/200 μL and administered to mice. Three days after En. faecalis administration, serotonin content in the colon tissue was evaluated. These experiments were independently performed twice (n = 4 and 6 in each group, respectively), with a resulting n = 10 per group.

Quantification of serotonin concentration in the colon tissue
Mice were euthanized by cervical dislocation under the anesthetization with Dormicum (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), Vetorphale (Astellas Pharma, Inc., Tokyo, Japan), and Domitor (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan). The entire length of the colon with fullthickness was washed with PBS to remove the luminal contents, sonicated in 10 mL of PBS using Branson model 250 (BRANSON, St. Louis, MO, USA), and stored at −25°C, until use. The colon lysate was centrifuged (21,500 × g, 4°C, 5 min) to remove tissue debris. Serotonin concentration was determined using Serotonin ELISA Kit (Enzo Lifescience, Farmingdale, NY, USA), according to the manufacturer's protocol. The serotonin ELISA Kit (Enzo Lifescience, Farmingdale, NY, USA) was used because the Serotonin ELISA Kit (Eagle Biosciences) used by Yano et al. 24 could not be purchased in Japan due to legal restrictions. Serotonin amounts were normalized based on the weight of the colonic tissue. Animal experiments were independently performed twice (n = 4 and 6 in each group, respectively), and for each experiment, the amount of serotonin was normalized by the average amount of serotonin in mice colonized with wild-type En. faecalis. The normalized values were assigned an arbitrary unit (a.u.) and the serotonin levels in the colonic tissue were expressed as serotonin (a.u.).

Analysis of Taar1 and Mao-b expression in mouse colon
Colon tissue from six-week-old female BALB/ cCrSlc mice was purchased from Japan SLC. The tissue was treated with RNAlater-ICE (Thermo Fisher Scientific) for 20 h at −20°C and used for RNA extraction. The frozen colon was placed into a tube with a stainless bead and disrupted using a Multi-Beads shocker (Yasui Kikai Co., Osaka, Japan). RNA extraction and on-column genomic DNA digestion were then performed using ISOSPIN Cell & Tissue RNA Kit (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 0.2 or 1 μg of RNA using PrimeScript™ RT reagent Kit (Perfect Real Time) (TaKaRa Bio). TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa Bio) was used for PCR, which was performed using 1 µL cDNA solution for samples that used 0.2 μg of RNA for cDNA synthesis and 0.2 μL cDNA solution for samples that used 1 μg of RNA for cDNA synthesis as a template. The PCR cycling conditions were 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The PCR products were electrophoresed with 3% agarose gel and visualized using ethidium bromide under ultraviolet light.

Statistical analysis
Statistical analyses were performed using SPSS software version 21 (IBM, Armonk, NY) and BellCurve for Excel (Social Survey Research Information Co., LTD.). Correlation between PEA production and aadc copy number was analyzed by Spearman's rank correlation test. Repeated measures one-way